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Kinetic analysis of red pigment and citrinin production by Monascusruberas a function of organic acid accumulation

Hassan Hajjaj, Philippe Blanc, Evelyne Groussac, Jean-Louis Uribelarrea, Gerard Goma,Pascal Loubiere*

Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, UMR-INRA 792, Institut National des Sciences Appliquees, 135 Avenue de Rangueil,

F-31077 Toulouse Cedex 04, France

Received 15 July 1999; received in revised form 8 June 2000; accepted 3 July 2000

Abstract

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruberproduced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of asecondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidlythroughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malateand succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, thisinhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate ortartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decreasein red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored inaerobic conditions. 2000 Elsevier Science Inc. All rights reserved.

Keywords:Filamentous fungus; Monascus;Pigments; Citrinin; Mycotoxin; Polyketide; Malate; Succinate; Dicarboxylic acid

1. Introduction

Filamentous fungi have important properties which playa significant role in the human lifestyle and in the environ-ment, by participating in the production of food and healthproducts, and in recycling of organic compounds in thebiosphere. Their biochemical potential and their adaptationto extreme life conditions in liquid media have been ex-ploited to produce molecules such as antibiotics (e.g., pen-icillin, cephalosporins), enzymes (-amylase, cellulase), or-ganic acids (e.g., citric acid) [1,2] and food colourants (e.g.,

Anka) [3].Monascusis a filamentous fungus which produces pig-ments used for red coloring of rice wine, soybean cheese,fish and red meat. These pigments are principally used in thesouth of China, Taiwan, Japan and Indonesia. This fungus istraditionally cultivated on solid media, rice grains or bread,

though such solid-state fermentation does not enable theenvironmental parameters to be controlled, and submergedcultures in natural or synthetic media have been developedrecently.

Monascus can produce yellow (monascin and ankafla-vin), orange (monascorubrin and rubropunctatin) and redpigments (monascorubramine and rubropunctamine). Thesepigments are not hydrosoluble, unstable in extreme pH (2and 14), and with respect to heat and to light [4]. Theseintracellular lipophilic pigments can react with aminegroups of proteins, amino acids, nucleic acids and amino

sugars, leading to formation of extracellular water-solublepigments [4,5,6]. The recent use of glutamic acid as bothnitrogen source and reactive entity to stimulate the extra-cellular accumulation of pigments has improved the effi-ciency of pigment production [7,8] and facilitates the for-mation of a glutamic acid-pigment complex [9]. Thepigment molecule consists of two parts condensed by ester-ification: a short-chain fatty acid (C6 or C8) and a hexa-ketide [10] synthesized by the polyketide pathway. Re-cently, it was shown that red pigment production is

* Corresponding author. Tel.: 33-5-61-55-94-38; fax: 33-5-61-55-94-00.

E-mail address: [email protected] (P. Loubiere).

www.elsevier.com/locate/enzmictecEnzyme and Microbial Technology 27 (2000) 619625

0141-0229/00/$ see front matter 2000 Elsevier Science Inc. All rights reserved.PII: S0141-0229(00)00260-X

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accompanied by the production of a mycotoxin, citrinin[11], synthesised by the polyketide pathway [12]. Synthesisis derived from a tetraketide precursor in Monascusratherthan a pentaketide as observed in Aspergillus terreus orPenicillium citrinum[13]. Citrinin has nephrotoxic and hep-atotoxic properties [14].

Despite a common biosynthetic pathway for the redpigment and citrinin, their production is differently affectedby nutritional and environmental parameters. Agitation andaeration are parameters which strongly influence growthand secondary metabolite production in M. ruber. In con-ditions of high oxygen supply, citrinin production was fa-vored, and moreover, a linear relationship was observedbetween the oxygen supply and the citrinin yield [15]. Incontrast, pigment synthesis increased with the oxygen sup-ply in an hyperbolic profile, illustrating that pigment pro-duction is inhibited in cultures under aerobic conditions.The growth ofM. ruberin a synthetic medium containingglucose and glutamate was associated with the co-produc-tion of L-malate and succinate.

In this study, the production of these dicarboxylic acidswas studied in relation to oxygen supply and secondarymetabolite production. The effect of the addition of theseacids on growth, and citrinin and red pigment productionwas also examined. It was concluded that dicarboxylic acidsweakly inhibited red pigment synthesis, but this effect couldnot account for the decrease in pigment production observedduring a culture, indicating that other inhibitory compoundswere produced.

2. Materials and methods

2.1. Micro-organism and growth conditions

The strain used was a strain ofMonascus ruber(classi-fied as ATCC 96218) obtained from anka, producing largequantities of pigment.

The culture medium was the chemically-defined mediumdeveloped in previous studies [6] and contained: glucose,20 g; monosodium glutamate (MSG), 5 g; K2HPO4, 5 g;KH2PO4,5 g; CaCl2,0.1 g; MgSO4.7H2O, 0.5 g; FeSO4.7H2O, 0.01 g; ZnSO4.7H2O, 0.01 g; MnSO4.H2O, 0.03 gper liter of distilled water. The initial pH of the medium wasadjusted to 6.5 with phosphoric acid (H3PO4), or potassiumhydroxide (KOH) when L-malic or succinic acid wereadded to the medium, as stated in the text.

The stock culture was maintained on Difco potato dex-trose agar (PDA). Spores were prepared by growth on PDAslants for 10 days at 30C, and were harvested and washedwith sterile water. A suspension of 108 spores was used toinoculate a 1l baffled Erlenmeyer flask containing 200 ml ofmedium. This inoculum was incubated at 30C for 2 daysand then transferred to 2 liters fermentors containing 1.3liters of synthetic medium.

The cultures were incubated at 30C, with different aer-

ation rates. The pH of the cultures was not regulated but thephosphate buffer avoided a significant variation of the pHthroughout the culture.

2.2. Analytical Methods

Fungal biomass was determined by gravimetric analysisafter filtration of cell samples through preweighed nylonfilters (45 mm diameter, 0.8 m porosity) and dried toconstant weight at 60C under partial vacuum (200 mmHg).

Glucose was measured using an autoanalyser YSI 200(YSI Inc., Yellow Springs, Ohio). Ethanol and acetic acidconcentrations were determined by gas chromatography us-ing a flame ionisation detector and a Poraplot Q column (25mm 0.53 mm) at 190C and N2 flow rate of 30 li-ters.min1. The concentration of malic acid and succinic

acid was analyzed by High-Pressure Liquid Chromatogra-phy (HPLC) (HP 1050; Hewlett Packard, Grenoble, France)equipped with an integrator (HP 3396A) and an automaticsampler (SP 8775; Spectra Physic France, Les Ulis, France).Detection was made at 210 nm with a variable-wave-length UV detector (Hewlett Packard, HP series 1050).The separation was achieved with an Aminex (HPX-87H Bio-Rad Chemical Division, Richmond, Calif.)column (300 by 7.8 mm), using the following operatingconditions: temperature, 50C; mobile phase, 5 mMH2SO4; flow rate, 0.5 ml/min.

The L-malic acid was assayed by spectrophotometric

measurement of variation in NADPH concentrations using amalic enzyme specific for L-malic acid. The reaction mix-ture contained phosphate buffer (pH 7.8, 100 mM), MgCl2(5 mM), NADP (0.6 mM) and malic enzyme (200 U/mgproteins).

Glutamic acid in the medium was quantified with anAminoQuant 1090 HPLC (Hewlett-Packard) after derivati-zation with ortho-phthalaldehyde in the presence of 3-mer-captopropionic acid, according to Hewlett-Packard proce-dure.

O2 and CO2 concentrations in the outflow gas weremeasured with a gas chromatograph (column of Porapak Q

molecular sieve 5 , 80100 mesh, 40C, helium flowrate 60 ml.min1).

The red pigment was spectrophotometrically determinedby measuring the absorbance of culture filtrate at 480 nm: 1unit O.D.480nmcorresponded to 15 mg.l

1 pigment [6], andthe average mass of the red pigment was 498 g.mol1.

Citrinin (molecular mass 250 g.mol1) was deter-mined by HPLC on a C18column using the following linearseparation gradient: water/methanol (80/20, v/v) to water/methanol (0/100, v/v) in 30 min. The flow rate was 0.8ml/min. The detector used was a Waters UV spectropho-tometer, and max 260 nm.

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3. Results and Discussion

3.1. Growth and metabolic behavior ofM. ruber in abatch-mode fermentation

A typical batch-mode fermentation time-course of M.

ruber,in a medium containing 20 g.l1

glucose and 5 g.l1

monosodium glutamate (MSG), a stirrer speed of 250 rpmand an aeration rate of 0.04 vvm (standard conditions formixing and aeration), is shown in Fig. 1. Glucose and MSGwere consumed simultaneously. Glucose was exhaustedfrom the medium after about 120 h of cultivation, while2 g.l1 MSG remained in the medium. The biomass con-centration at the time of glucose exhaustion was 5.0 g.l1,

corresponding to a biomass yield relative to glucose con-sumption of 0.24 g.g1. The red pigment was producedthroughout the fermentation and reached a concentration of87 mg.l1, corresponding to an OD480nmvalue of 5.8. An-other secondary metabolite, citrinin, was produced afterabout 50 h cultivation and attained 36 mg.l1 by the end ofthe fermentation. As regards fermentation products, ethanol,malate, succinate and CO2 were produced in significantamounts, while acetate and fumarate were produced at avery low level. Neither citrate nor isocitrate were detected inthe fermentation broth. As regards organic acid production,

M. ruberproduced specifically L-malic acid throughout theentire fermentation, in 2-fold higher concentrations thansuccinic acid. The pH of the culture medium decreasedfrom 6.7 to 6.1. Oxygen uptake rate and carbon dioxideproduction rate each increased throughout the fermenta-tion (Fig 1C), but the oxygen level remained sufficiently

high to avoid anoxic growth conditions.

3.2. Specific rates of growth, red pigment and citrinin

production as a function of aeration

During the fermentation period of about 120 h for thereference culture with 0.04 vvm aeration, the specific ratesof growth, of substrate consumption, and of ethanol, malate,succinate, citrinin or red pigment production, increased dur-ing the initial 15% of the culture duration, and decreasedthereafter (Fig 2A). The accelerating growth phase has beenrelated to carbon dioxide limitation, since supplying CO2-enriched air enabled the specific growth rate to rapidly reachthe maximal value [8]. The specific rate values were max-imal in the middle part of the time-cultivation and corre-sponded to a relatively small fraction of glucose consump-tion, i.e. a low value of the fermentation progress expressedas the fraction of glucose consumed (Fig 2). The maximalvalues observed for the specific growth rate, glucose andMSG substrate consumption rate, and red pigment and cit-rinin production rate, were as follows: max 0.04 h

1; qglu 0.15 g.g1.h1; qMSG 0.015 g.g

1.h1; p red pig-ment 1.52 mg.g1.h1; p citrinin 0.3 mg.g1.h1.The early and simultaneous decrease in the specific rates ofred pigment and citrinin production was unusual in the light

of the current knowledge on secondary metabolite produc-tion, and was more representative of kinetic profiles asso-ciated with primary metabolite production.

In the more aerated culture (0.4 vvm, Fig 2B), the pro-files of the specific rates of growth, red pigment and citrininproduction were different. The specific growth rate in-

creased between 0.05 and 0.32 of fermentation progress toreach a maximal value of 0.035 h1, and decreased there-after. The specific rate of citrinin production increased at theonset of fermentation and was maintained at the maximalvalue during the entire time-course of the culture. Thisprofile was characteristic of the production of a secondarymetabolite. The specific rate of red pigment productionincreased in the first part of the culture, and then decreasedearly as previously observed, while the growth rate contin-ued to increase.

When the culture was strongly aerated (2 vvm, Fig 2C),the growth rate increased rapidly to reach a value of 0.036h1 at a fermentation progress of about 0.1, and decreasedthereafter. A similar profile was observed for the specificrate of red pigment production, the maximal value being1.39 mg.g1.h1. On the other hand, the specific rate ofcitrinin production increased and was maintained at a highvalue (0.68 mg.g1.h1) throughout the culture.

As previously observed [15] increasing aeration im-proved citrinin production, since not only had the maximalspecific rate reached during the culture increased, but alsothis maximal value was maintained for a longer period. Thisobservation was explained by the fact that oxygen wasconsumed in the citrinin production pathway. In the leastaerated culture, it can be proposed that the decreased spe-

cific rate of citrinin production observed during the culturewas due to a limited oxygen availability, as illustrated by arespiro-fermentative metabolism with production of a highethanol concentration (3.5 g.l1). On the other hand, thedecrease in pigment production rate observed whatever theaeration level, might be related to the variable concentrationof a component of the medium, most probably a product ofthe fermentation whose accumulation in the medium spe-cifically inhibited red pigment production. Only four fer-mentation products excreted in significant amounts (CO2,ethanol, malate and succinate) were identified. Among theidentified products, CO2has been shown previously to haveno effect on production [8], since the specific rate of pig-ment production was not modified even when supplying themedium with CO2-enriched air. Ethanol has been used as acarbon substrate for red pigment synthesis [9,16] and doesnot diminish production. Therefore, the possibility thatmalate or succinate produced during the culture, could beresponsible for the decrease in pigment synthesis was ex-amined.

Malic and succinic acids were rapidly accumulated at theonset of the culture, in the phase where the growth rate andthe specific rates of red pigment and citrinin increased (Fig2). The second phase in which the rate of accumulation ofthe organic acids was reduced correlated with the decrease

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Fig. 1. Fermentation time-course forM. rubergrowing on glucose (20 g.l1) at an aeration rate of 0.04 vvm and an agitation speed of 250 rpm. Abbreviations:S glucose; X biomass; MSG monosodium glutamate; rCO2 rate of carbon dioxide production; rO2 rate of oxygen consumption; %pO2 oxygenpartial pressure in percentage.


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Fig. 2. Evolution of the specific growth rate (), of the specific rate of citrinin production (p citrinin), of the specific rate of red pigment production (p redpigment), and of malic acid concentration, as a function of the fermentation progress relative to the glucose consumed (1-S/So), during cultures ofM. ruberat an agitation speed of 250 rpm, and aeration rates of 0.04 vvm (A), 0.4 vvm (B), and 2 vvm (C).


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in both the growth rate and the specific rate of red pigmentproduction. The transition between these two phases oc-curred when the malate and succinate concentration reacheda value of about 0.5 and 0.25 g.l1 respectively, whatever theaeration level. This apparent correlation between the dicar-boxylic acid accumulation and the decrease in the rate of red

pigment production could indicate that these acids were di-rectly responsible for the inhibition of red pigment synthesis.

3.3. Effect of added L-malate and succinate on growth

and red pigment production

M. ruber poorly assimilated L-malate as sole carbonsource since 2 g.l1 of malate were totally consumed inabout ten weeks. In this condition, no red pigment wasproduced. On the other hand, no growth was observed onfumarate or succinate as sole carbon source.

Since organic acid (i.e., L-malate and succinate) accu-mulated in the fermentation broth were suspected as havinga detrimental effect on pigment production, the effect ofadding dicarboxylic acid to the initial growth medium wasexamined with respect to both growth and secondary me-tabolism ofM. ruber.Fermentations were carried out in thepresence of L-malate or succinate concentrations rangingfrom 0 to 4 g.l1, and the initial pH of all the culturesperformed was 6.5.

During these cultures performed with glucose as sub-strate, the added organic acids were not consumed, while onthe other hand, L-malate and succinate were produced atconcentrations of about 1 g.l1 and 0.45 g.l1 respectively(data not shown). The maximal specific rates of growth and

of glucose and glutamate consumption varied slightly withthe concentration of added acids. The final biomass concen-tration did not vary significantly with the L-malate or thesuccinate concentration, indicating that the biomass yieldremained constant.

Red pigment production decreased with the concentra-tion of added dicarboxylic acid. The final concentration ofred pigment was 13% and 27% lower in presence of 2 and4 g.l1 of L-malate respectively, compared to the controlwithout added malate (Fig 3A). The effect of succinate wasquite similar since the addition of 4 g.l1 led to a decreasein the red pigment production of about 20% (Fig 3B). Incontrast, neither the profile of citrinin accumulation nor thefinal citrinin concentration were affected by the presence ofthese acids whatever the concentration tested, indicatingthat only the red pigment production was affected by thepresence of dicarboxylic acids.

The addition in the culture medium of other dicarboxylicacids such as D-malate, fumarate, L-tartrate or D-tartrate ata concentration of 2 g.l1 had a similar effect on red pig-ment production as that observed in the presence of L-malateor succinate, while citrinin production was unchanged. On theother hand, the accumulation of monocarboxylic acid such asacetate at concentrations of up to 10 g.l1 had no detrimentaleffect on the pigment production.

Since all the dicarboxylic acids tested and not only themetabolisable acids had an inhibitory effect on the pigmentproduction, it may be proposed that the effect was notrelated to the consumption of the acids, or to the interme-diates of the TCA cycle. Moreover, since a common meta-bolic pathway, the polyketide pathway, is involved in thesynthesis of both the pigment and citrinin, it may be con-cluded that the action of dicarboxylic acid must be localizedin reactions specific to pigment synthesis. The only differ-ence in the metabolic pathways is that pigment productioninvolves a condensation step by esterification between thechromophore (the polyketide fraction) and a fatty acid chain(C6 or C8) [10]. Hence, it would appear that the negativeeffect of dicarboxylic acid on pigment production probablyinvolves fatty acid synthesis.

Despite the observed negative effect of the dicarboxylicacids on red pigment production, excretion of these acidsdid not have a significant effect during the culture of M.ruber. The concentration of L-malate and succinate ob-

Fig. 3. Red pigment production with various (A) L-malic acid and (B)succinic acid concentrations added to the culture, during growth of M.ruberon glucose.


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served in the culture when the rate of production of pigmentbegan to decrease was about 0.5 and 0.25 g.l1 respectively,and the production virtually stopped when about 1 g.l1

L-malate and 0.5 g.l1 succinate were accumulated in themedium. However, these last concentration values couldtheoretically be responsible at most for about 1015% for

the inhibition of pigment production.From these results, it seems that unidentified compoundsproduced during the fermentation must have an importantinhibitory effect on red pigment production. It is known thatpolyketide synthases are unstable enzymes in vivo [17], dueto the production of intracellular proteases [18]. It has beenproposed recently that the synthesis of proteases enhancingthe decay of pigment synthase was induced in the presenceof L-leucine [5], though these proteases played a minor rolein the decreased pigment synthesis. Our culture medium didnot contain L-leucine, but since very little is known aboutthe protease induction, the occurrence of this phenomenonin our strain and in the presence of glutamate cannot betotally neglected. The inhibitory compound produced dur-ing our culture was favored in aerobic conditions, since thedecrease in pigment synthesis appeared earlier and wasmore pronounced in strongly aerated cultures. This productof aerobic metabolism and its mode of action on the pig-ment synthesis remain to be elucidated. To date, the opti-mization of the ratio of red pigment to citrinin productionrequires a tight compromise between a weak aeration inwhich oxygen supply can limit the pigment production, and astrong aeration which can favour the production of inhibitorycompounds. The nature of the amino acids present in theculture medium should be another important parameter sus-

ceptible to play a role in the synthesis of potentially inhibitorycompounds, and work is in progress in the laboratory to studythis effect on the pigment and citrinin synthesis.

Acknowledgments

H. Hajjaj grateful acknowledges INRA (Institut Nationalde la Recherche Agronomique) France for his financialsupport. We thank Pr. J.M. Francois and Dr. N.D. Lindleyfor useful discussions.

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